Method for detecting HTRAN polynucleotides

ABSTRACT

The present invention provides a human transcription factor (HTRAN) and polynucleotides which identify and encode HTRAN. The invention also provides expression vectors and host cells, agonists, antibodies, or antagonists. The invention provides methods for treating diseases associated with expression of HTRAN.

This application is a divisional application of U.S. application Ser.No. 09/059,520, filed Apr. 13, 1998, which is a divisional of U.S.application Ser. No. 08/843,993, filed Apr. 17, 1997, issued Apr. 14,1998, as U.S. Pat. No. 5,739,010.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences of anovel human transcription factor and to the use of these sequences inthe diagnosis, prevention, and treatment of cancer, arthritis, anddevelopmental disorders.

BACKGROUND OF THE INVENTION

Regulation of gene transcription is the primary process by which a cellcontrols the appropriate expression of the multitude of genes necessaryfor growth and differentiation. The selective expression of genes atappropriate times is highly specialized in cells of multicellularorganisms and permits the cells to perform "housekeeping" functions andrespond to changes in their environment. These changes involveextracellular signals from a variety of sources such as hormones,neurotransmitters, and growth and differentiation factors.

Gene transcription is controlled by proteins termed regulators of genetranscription (RGT). RGTs act by binding to a short segment of DNA(transcription control element, TCE) located near the site oftranscription initiation. Binding of an RGT to the target TCE activatestranscription of the gene. RGTs contain a variety of structural motifsthat, alone or in combination with one another, permit them to recognizeand bind to the wide variety of TCEs.

One group of RGTs, the TFIIIA subclass of zinc-finger proteins, ischaracterized by an amino acid motif (a cysteine followed by two to fouramino acids, a cysteine, twelve amino acids, a histidine, three to fouramino acids, and a histidine) that interacts with zinc ions. Thecarboxyl terminus of the TFIIIA proteins has three of these "zincfinger" motifs and specifically binds to DNA fragments containing aCACCC pattern. The amino-terminal portion of the TFIIIA proteins isproline and serine-rich and can function as a transcriptional activator.

TFIIIA proteins are often important for the proper differentiation oftissues in which they are expressed. For example, the erythroidKruppel-like factor (EKLF) is a TFIIIA subclass zinc-finger protein thatis expressed in erythroid cells and regulates the B-globin gene. Loss offunctional EKLF in mice results in lethal anemia since B-globin is notsynthesized (Perkins A. C. et al (1995) Nature 375: 318-322). Anothermember of this class of proteins, WT-1, is expressed duringembryogenesis in the kidney and genital tissues (Pritchard-Jones K. etal. (1990) Nature 346: 194-197). In mice loss of functional WT-1 proteinresults in failure of the kidney and gonads to form (Kreidberg J. A. etal. (1993) Cell 74:679-691). Mouse BKLF has also been characterized as aerythroid Kruppel-like transcription factor (GI 1244515).

Discovery of proteins related to mouse BKLF and the polynucleotidesencoding them satisfies a need in the art by providing new compositionsuseful in diagnosis, prevention, and treatment of cancer, arthritis, anddevelopmental disorders.

SUMMARY OF THE INVENTION

The present invention features a novel human transcription factorhereinafter designated HTRAN and characterized as having chemical andstructural similarity to mouse BKLF and other Kruppel-like transcriptionfactors.

Accordingly, the invention features a substantially purified HTRAN whichhas the amino acid sequence shown in SEQ ID NO:1.

One aspect of the invention features isolated and substantially purifiedpolynucleotides that encode HTRAN. In a particular aspect, thepolynucleotide is the nucleotide sequence of SEQ ID NO:2.

The invention also relates to a polynucleotide sequence comprising thecomplement of SEQ ID NO:2 or variants thereof. In addition, theinvention features polynucleotide sequences which hybridize understringent conditions to SEQ ID NO:2.

The invention additionally features nucleic acid sequences encodingfragments, antisense molecules thereof, and expression vectors and hostcells comprising polynucleotides that encode HTRAN. The presentinvention also features antibodies which bind specifically to HTRAN, andpharmaceutical compositions comprising substantially purified HTRAN. Theinvention also features the use of agonists and antagonists of HTRAN.The invention also features a method for producing HTRAN using the hostcell and methods for treating developmental disorders by administeringHTRAN. In addition, the invention features methods for treating cancerand arthritis by administering an antagonist to HTRAN.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, 1C, 1D, and 1E show the amino acid sequence (SEQ ID NO:1)and nucleic acid sequence (SEQ ID NO:2) of HTRAN. The alignment wasproduced using MacDNASIS PRO™ software (Hitachi Software EngineeringCo., Ltd., San Bruno, Calif.).

FIGS. 2A and 2B show the amino acid sequence alignments among HTRAN (SEQID NO:1), mouse BKLF (GI 1244515; SEQ ID NO: 3), and human BTEB2 (GI303597; SEQ ID NO:4). The alignment was produced using the multisequencealignment program of DNASTAR™ software (DNASTAR Inc, Madison Wis.).

FIG. 3 shows the hydrophobicity plot (MacDNASIS PRO software) for HTRAN,SEQ ID NO:1; the positive X axis reflects amino acid position, and thenegative Y axis, hydrophobicity.

FIG. 4 shows the hydrophobicity plot for mouse BKLF (SEQ ID NO:3).

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms "a", "an", and "the" include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to "ahost cell" includes a plurality of such host cells, reference to the"antibody" is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

DEFINITIONS

"Nucleic acid sequence" as used herein refers to an oligonucleotide,nucleotide, or polynucleotide, and fragments or portions thereof, and toDNA or RNA of genomic or synthetic origin which may be single- ordouble-stranded, and represent the sense or antisense strand. Similarly,"amino acid sequence" as used herein refers to an oligopeptide, peptide,polypeptide, or protein sequence, and fragments or portions thereof, andto naturally occurring or synthetic molecules.

Where "amino acid sequence" is recited herein to refer to an amino acidsequence of a naturally occurring protein molecule, "amino acidsequence" and like terms, such as "polypeptide" or "protein" are notmeant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

"Peptide nucleic acid", as used herein, refers to a molecule whichcomprises an oligomer to which an amino acid residue, such as lysine,and an amino group have been added. These small molecules, alsodesignated anti-gene agents, stop transcript elongation by binding totheir complementary strand of nucleic acid (Nielsen, P. E. et al. (1993)Anticancer Drug Des. 8:53-63).

HTRAN, as used herein, refers to the amino acid sequences ofsubstantially purified HTRAN obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant.

"Consensus", as used herein, refers to a nucleic acid sequence which hasbeen resequenced to resolve uncalled bases, or which has been extendedusing XL-PCR™ (Perkin Elmer, Norwalk, Conn.) in the 5' and/or the 3'direction and resequenced, or which has been assembled from theoverlapping sequences of more than one Incyte clone using the GELVIEW™Fragment Assembly system (GCG, Madison, Wis.), or which has been bothextended and assembled.

A "variant" of HTRAN, as used herein, refers to an amino acid sequencethat is altered by one or more amino acids. The variant may have"conservative" changes, wherein a substituted amino acid has similarstructural or chemical properties, e.g., replacement of leucine withisoleucine. More rarely, a variant may have "nonconservative" changes,e.g., replacement of a glycine with a tryptophan. Similar minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

A "deletion", as used herein, refers to a change in either amino acid ornucleotide sequence in which one or more amino acid or nucleotideresidues, respectively, are absent.

An "insertion" or "addition", as used herein, refers to a change in anamino acid or nucleotide sequence resulting in the addition of one ormore amino acid or nucleotide residues, respectively, as compared to thenaturally occurring molecule.

A "substitution", as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively.

The term "biologically active", as used herein, refers to a proteinhaving structural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, "immunologically active" refers to thecapability of the natural, recombinant, or synthetic HTRAN, or anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

The term "agonist", as used herein, refers to a molecule which, whenbound to HTRAN, causes a change in HTRAN which modulates the activity ofHTRAN. Agonists may include proteins, nucleic acids, carbohydrates, orany other molecules which bind to HTRAN.

The terms "antagonist" or "inhibitor", as used herein, refer to amolecule which, when bound to HTRAN, blocks or modulates the biologicalor immunological activity of HTRAN. Antagonists and inhibitors mayinclude proteins, nucleic acids, carbohydrates, or any other moleculeswhich bind to HTRAN.

The term "modulate", as used herein, refers to a change or an alterationin the biological activity of HTRAN. Modulation may be an increase or adecrease in protein activity, a change in binding characteristics, orany other change in the biological, functional or immunologicalproperties of HTRAN.

The term "mimetic", as used herein, refers to a molecule, the structureof which is developed from knowledge of the structure of HTRAN orportions thereof and, as such, is able to effect some or all of theactions of transcription factor-like molecules.

The term "derivative", as used herein, refers to the chemicalmodification of a nucleic acid encoding HTRAN or the encoded HTRAN.Illustrative of such modifications would be replacement of hydrogen byan alkyl, acyl, or amino group. A nucleic acid derivative would encode apolypeptide which retains essential biological characteristics of thenatural molecule.

The term "substantially purified", as used herein, refers to nucleic oramino acid sequences that are removed from their natural environment,isolated or separated, and are at least 60% free, preferably 75% free,and most preferably 90% free from other components with which they arenaturally associated.

"Amplification" as used herein refers to the production of additionalcopies of a nucleic acid sequence and is generally carried out usingpolymerase chain reaction (PCR) technologies well known in the art(Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y.).

The term "hybridization", as used herein, refers to any process by whicha strand of nucleic acid binds with a complementary strand through basepairing.

The term "hybridization complex", as used herein, refers to a complexformed between two nucleic acid sequences by virtue of the formation ofhydrogen bonds between complementary G and C bases and betweencomplementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀ t or R₀ tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,membranes, filters, chips, pins or glass slides to which cells have beenfixed for in situ hybridization).

The terms "complementary" or "complementarity", as used herein, refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base-pairing. For example, for the sequence"A-G-T" binds to the complementary sequence "T-C-A". Complementaritybetween two single-stranded molecules may be "partial", in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between nucleic acids strands.

The term "homology", as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence is one that atleast partially inhibits an identical sequence from hybridizing to atarget nucleic acid; it is referred to using the functional term"substantially homologous." The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (i.e., the hybridization) of a completely homologoussequence or probe to the target sequence under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target sequence which lacks even apartial degree of complementarity (e.g., less than about 30% identity);in the absence of non-specific binding, the probe will not hybridize tothe second non-complementary target sequence.

As known in the art, numerous equivalent conditions may be employed tocomprise either low or high stringency conditions. Factors such as thelength and nature (DNA, RNA, base composition) of the sequence, natureof the target (DNA, RNA, base composition, presence in solution orimmobilization, etc.), and the concentration of the salts and othercomponents (e.g., the presence or absence of formamide, dextran sulfateand/or polyethylene glycol) are considered and the hybridizationsolution may be varied to generate conditions of either low or highstringency different from, but equivalent to, the above listedconditions.

The term "stringent conditions", as used herein, is the "stringency"which occurs within a range from about Tm-5° C. (5° C. below the meltingtemperature (Tm) of the probe) to about 20° C. to 25° C. below Tm. Aswill be understood by those of skill in the art, the stringency ofhybridization may be altered in order to identify or detect identical orrelated polynucleotide sequences.

The term "antisense", as used herein, refers to nucleotide sequenceswhich are complementary to a specific DNA or RNA sequence. The term"antisense strand" is used in reference to a nucleic acid strand that iscomplementary to the "sense" strand. Antisense molecules may be producedby any method, including synthesis by ligating the gene(s) of interestin a reverse orientation to a viral promoter which permits the synthesisof a complementary strand. Once introduced into a cell, this transcribedstrand combines with natural sequences produced by the cell to formduplexes. These duplexes then block either the further transcription ortranslation. In this manner, mutant phenotypes may be generated. Thedesignation "negative" is sometimes used in reference to the antisensestrand, and "positive" is sometimes used in reference to the sensestrand.

The term "portion", as used herein, with regard to a protein (as in "aportion of a given protein") refers to fragments of that protein. Thefragments may range in size from four amino acid residues to the entireamino acid sequence minus one amino acid. Thus, a protein "comprising atleast a portion of the amino acid sequence of SEQ ID NO:1" encompassesthe full-length human HTRAN and fragments thereof.

"Transformation", as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the host cell being transformedand may include, but is not limited to, viral infection,electroporation, lipofection, and particle bombardment. Such"transformed" cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

The term "antigenic determinant", as used herein, refers to that portionof a molecule that makes contact with a particular antibody (i.e., anepitope). When a protein or fragment of a protein is used to immunize ahost animal, numerous regions of the protein may induce the productionof antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

The terms "specific binding" or "specifically binding", as used herein,in reference to the interaction of an antibody and a protein or peptide,mean that the interaction is dependent upon the presence of a particularstructure (i.e., the antigenic determinant or epitope) on the protein;in other words, the antibody is recognizing and binding to a specificprotein structure rather than to proteins in general. For example, if anantibody is specific for epitope "A", the presence of a proteincontaining epitope A (or free, unlabeled A) in a reaction containinglabeled "A" and the antibody will reduce the amount of labeled A boundto the antibody.

The term "sample", as used herein, is used in its broadest sense. Abiological sample suspected of containing nucleic acid encoding HTRAN orfragments thereof may comprise a cell, chromosomes isolated from a cell(e.g., a spread of metaphase chromosomes), genomic DNA (in solution orbound to a solid support such as for Southern analysis), RNA (insolution or bound to a solid support such as for northern analysis),cDNA (in solution or bound to a solid support), an extract from cells ora tissue, and the like.

The term "correlates with expression of a polynucleotide", as usedherein, indicates that the detection of the presence of ribonucleic acidthat is similar to SEQ ID NO:2 by northern analysis is indicative of thepresence of mRNA encoding HTRAN in a sample and thereby correlates withexpression of the transcript from the polynucleotide encoding theprotein.

"Alterations" in the polynucleotide of SEQ ID NO: 2, as used herein,comprise any alteration in the sequence of polynucleotides encodingHTRAN including deletions, insertions, and point mutations that may bedetected using hybridization assays. Included within this definition isthe detection of alterations to the genomic DNA sequence which encodesHTRAN (e.g., by alterations in the pattern of restriction fragmentlength polymorphisms capable of hybridizing to SEQ ID NO:2), theinability of a selected fragment of SEQ ID NO: 2 to hybridize to asample of genomic DNA (e.g., using allele-specific oligonucleotideprobes), and improper or unexpected hybridization, such as hybridizationto a locus other than the normal chromosomal locus for thepolynucleotide sequence encoding HTRAN (e.g., using fluorescent in situhybridization [FISH] to metaphase chromosomes spreads).

As used herein, the term "antibody" refers to intact molecules as wellas fragments thereof, such as Fab F(ab')₂, and Fv, which are capable ofbinding the epitopic determinant. Antibodies that bind HTRANpolypeptides can be prepared using intact polypeptides or fragmentscontaining small peptides of interest as the immunizing antigen. Thepolypeptide or peptide used to immunize an animal can be derived fromthe transition of RNA or synthesized chemically, and can be conjugatedto a carrier protein, if desired. Commonly used carriers that arechemically coupled to peptides include bovine serum albumin andthyroglobulin. The coupled peptide is then used to immunize the animal(e.g., a mouse, a rat, or a rabbit).

The term "humanized antibody", as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

THE INVENTION

The invention is based on the discovery of a novel human transcriptionfactor, (HTRAN), the polynucleotides encoding HTRAN, and the use ofthese compositions for the diagnosis, prevention, or treatment ofcancer, arthritis, and developmental disorders.

Nucleic acids encoding the human HTRAN of the present invention werefirst identified in Incyte Clone 727885 from the human knee synovialmembrane tissue cDNA library (SYNOOAT01) through a computer-generatedsearch for amino acid sequence alignments. A consensus sequence, SEQ IDNO:2, was derived from the assembled and/or extended nucleic acidsequences of Incyte clones 727885 (SYNOOAT01) and 13286 (THP1PLB01).

In one embodiment, the invention encompasses a polypeptide comprisingthe amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A, 1B, 1C,1D, and 1E. HTRAN is 345 amino acids in length and has a potentialN-glycosylation site at asparagine residue 175. HTRAN has threeconsensus zinc finger motifs that begin at cysteine residues 262, 292,and 322. Like other Kruppel-like transcription factors, the region ofHTRAN amino-terminal to the zinc finger motifs is very rich in prolineand serine residues (frequency of 31%). HTRAN has chemical andstructural homology with mouse BKLF (GI 1244515; SEQ ID NO: 3; FIGS. 2Aand 2B). In particular, HTRAN and mouse BKLF share 96% identity. Asillustrated by FIGS. 3 and 4, HTRAN and mouse BKLF have rather similarhydrophobicity plots. Northern analysis revealed the expression of mRNAencoding HTRAN in the knee synovium of an arthritis patient and intumors taken from the colon, lung, and bladder of cancer patients.

The invention also encompasses HTRAN variants. A preferred HTRAN variantis one having at least 80%, and more preferably 90%, amino acid sequenceidentity to the HTRAN amino acid sequence (SEQ ID NO:1). A mostpreferred HTRAN variant is one having at least 95% amino acid sequenceidentity to SEQ ID NO:1.

The invention also encompasses polynucleotides which encode HTRAN.Accordingly, any nucleic acid sequence which encodes the amino acidsequence of HTRAN can be used to generate recombinant molecules whichexpress HTRAN. In a particular embodiment, the invention encompasses thepolynucleotide comprising the nucleic acid sequence of SEQ ID NO:2 asshown in FIGS. 1A, 1B, 1C, 1D, and 1E.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of nucleotide sequencesencoding HTRAN, some bearing minimal homology to the nucleotidesequences of any known and naturally occurring gene, may be produced.Thus, the invention contemplates each and every possible variation ofnucleotide sequence that could be made by selecting combinations basedon possible codon choices. These combinations are made in accordancewith the standard triplet genetic code as applied to the nucleotidesequence of naturally occurring HTRAN, and all such variations are to beconsidered as being specifically disclosed.

Although nucleotide sequences which encode HTRAN and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring HTRAN under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding HTRAN or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding HTRAN and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

The invention also encompasses production of DNA sequences, or portionsthereof, which encode HTRAN and its derivatives, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents that are well known in the art at the time of the filing ofthis application. Moreover, synthetic chemistry may be used to introducemutations into a sequence encoding HTRAN or any portion thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed nucleotide sequences, and inparticular, those shown in SEQ ID NO:2, under various conditions ofstringency. Hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid binding complex or probe, as taughtin Wahl, G. M. and S. L. Berger (1987; Methods Enzymol. 152:399-407) andKimmel, A. R. (1987; Methods Enzymol. 152:507-511), and may be used at adefined stringency.

Altered nucleic acid sequences encoding HTRAN which are encompassed bythe invention include deletions, insertions, or substitutions ofdifferent nucleotides resulting in a polynucleotide that encodes thesame or a functionally equivalent HTRAN. The encoded protein may alsocontain deletions, insertions, or substitutions of amino acid residueswhich produce a silent change and result in a functionally equivalentHTRAN. Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological activity of HTRAN is retained. For example, negativelycharged amino acids may include aspartic acid and glutamic acid;positively charged amino acids may include lysine and arginine; andamino acids with uncharged polar head groups having similarhydrophilicity values may include leucine, isoleucine, and valine;glycine and alanine; asparagine and glutamine; serine and threonine;phenylalanine and tyrosine.

Also included within the scope of the present invention are alleles ofthe genes encoding HTRAN. As used herein, an "allele" or "allelicsequence" is an alternative form of the gene which may result from atleast one mutation in the nucleic acid sequence. Alleles may result inaltered mRNAs or polypeptides whose structure or function may or may notbe altered. Any given gene may have none, one, or many allelic forms.Common mutational changes which give rise to alleles are generallyascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

Methods for DNA sequencing which are well known and generally availablein the art may be used to practice any embodiments of the invention. Themethods may employ such enzymes as the Klenow fragment of DNA polymeraseI, Sequenase® (US Biochemical Corp, Cleveland, Ohio), Taq polymerase(Perkin Elmer), thermostable T7 polymerase (Amersham, Chicago, Ill.), orcombinations of recombinant polymerases and proofreading exonucleasessuch as the ELONGASE Amplification System marketed by Gibco BRL(Gaithersburg, Md.). Preferably, the process is automated with machinessuch as the Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.), PeltierThermal Cycler (PTC200; MJ Research, Watertown, Mass.) and the ABI 377DNA sequencers (Perkin Elmer).

The nucleic acid sequences encoding HTRAN may be extended utilizing apartial nucleotide sequence and employing various methods known in theart to detect upstream sequences such as promoters and regulatoryelements. For example, one method which may be employed,"restriction-site" PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to linker sequence and a primer specific to the knownregion. The amplified sequences are then subjected to a second round ofPCR with the same linker primer and another specific primer internal tothe first one. Products of each round of PCR are transcribed with anappropriate RNA polymerase and sequenced using reverse transcriptase.

Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed using OLIGO4.06 Primer Analysis software (National Biosciences Inc., Plymouth,Minn.), or another appropriate program, to be 22-30 nucleotides inlength, to have a GC content of 50% or more, and to anneal to the targetsequence at temperatures about 68°-72° C. The method uses severalrestriction enzymes to generate a suitable fragment in the known regionof a gene. The fragment is then circularized by intramolecular ligationand used as a PCR template.

Another method which may be used is capture PCR which involves PCRamplification of DNA fragments adjacent to a known sequence in human andyeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1:111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown portion of the DNA moleculebefore performing PCR.

Another method which may be used to retrieve unknown sequences is thatof Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PromoterFinder™libraries to walk in genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable, in that they will contain moresequences which contain the 5' regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into the 5' and 3'non-transcribed regulatory regions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentfluorescent dyes (one for each nucleotide) which are laser activated,and detection of the emitted wavelengths by a charge coupled devicecamera. Output/light intensity may be converted to electrical signalusing appropriate software (e.g. Genotyper™ and Sequence Navigator™,Perkin Elmer) and the entire process from loading of samples to computeranalysis and electronic data display may be computer controlled.Capillary electrophoresis is especially preferable for the sequencing ofsmall pieces of DNA which might be present in limited amounts in aparticular sample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode HTRAN, or fusion proteins or functionalequivalents thereof, may be used in recombinant DNA molecules to directexpression of HTRAN in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and these sequences may be used to clone and expressHTRAN.

As will be understood by those of skill in the art, it may beadvantageous to produce HTRAN-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce a recombinant RNAtranscript having desirable properties, such as a half-life which islonger than that of a transcript generated from the naturally occurringsequence.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alter HTRANencoding sequences for a variety of reasons, including but not limitedto, alterations which modify the cloning, processing, and/or expressionof the gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, or introduce mutations, and so forth.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding HTRAN may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of HTRAN activity, it may be useful toencode a chimeric HTRAN protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the HTRAN encoding sequence and theheterologous protein sequence, so that HTRAN may be cleaved and purifiedaway from the heterologous moiety.

In another embodiment, sequences encoding HTRAN may be synthesized, inwhole or in part, using chemical methods well known in the art (seeCaruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223,Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of HTRAN, or a portion thereof.For example, peptide synthesis can be performed using varioussolid-phase techniques (Roberge, J. Y. et al. (1995) Science269:202-204) and automated synthesis may be achieved, for example, usingthe ABI 431A Peptide Synthesizer (Perkin Elmer).

The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, WH Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of HTRAN, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

In order to express a biologically active HTRAN, the nucleotidesequences encoding HTRAN or functional equivalents, may be inserted intoappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding HTRAN andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

A variety of expression vector/host systems may be utilized to containand express sequences encoding HTRAN. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

The "control elements" or "regulatory sequences" are thosenon-translated regions of the vector--enhancers, promoters, 5' and 3'untranslated regions--which interact with host cellular proteins tocarry out transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the Bluescript® phagemid (Stratagene,LaJolla, Calif.) or pSport1™ plasmid (Gibco BRL) and the like may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding HTRAN,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for HTRAN. For example, when largequantities of HTRAN are needed for the induction of antibodies, vectorswhich direct high level expression of fusion proteins that are readilypurified may be used. Such vectors include, but are not limited to, themultifunctional E. coli cloning and expression vectors such asBluescript® (Stratagene), in which the sequence encoding HTRAN may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (supra)and Grant et al. (1987) Methods Enzymol. 153:516-544.

In cases where plant expression vectors are used, the expression ofsequences encoding HTRAN may be driven by any of a number of promoters.For example, viral promoters such as the 35S and 19S promoters of CaMVmay be used alone or in combination with the omega leader sequence fromTMV (Takamatsu, N. (1987) EMBO J. 6:307-311). Alternatively, plantpromoters such as the small subunit of RUBISCO or heat shock promotersmay be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R.et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) ResultsProbl. Cell Differ. 17:85-105). These constructs can be introduced intoplant cells by direct DNA transformation or pathogen-mediatedtransfection. Such techniques are described in a number of generallyavailable reviews (see, for example, Hobbs, S. or Murry, L. E. in McGrawHill Yearbook of Science and Technology (1992) McGraw Hill, New York,N.Y.; pp. 191-196).

An insect system may also be used to express HTRAN. For example, in onesuch system, Autographa californica nuclear polyhedrosis virus (AcNPV)is used as a vector to express foreign genes in Spodoptera frugiperdacells or in Trichoplusia larvae. The sequences encoding HTRAN may becloned into a non-essential region of the virus, such as the polyhedringene, and placed under control of the polyhedrin promoter. Successfulinsertion of HTRAN will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which HTRAN may be expressed (Engelhard, E. K. et al. (1994)Proc. Nat. Acad. Sci. 91:3224-3227).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, sequences encoding HTRAN may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain a viable virus which iscapable of expressing HTRAN in infected host cells (Logan, J. and Shenk,T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding HTRAN. Such signals include the ATGinitiation codon and adjacent sequences. In cases where sequencesencoding HTRAN, its initiation codon, and upstream sequences areinserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a portion thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers which are appropriate for theparticular cell system which is used, such as those described in theliterature (Scharf, D. et al. (1994) Results Probl. Cell Differ.20:125-162).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a "prepro" form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, HEK293, andW138, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressHTRAN may be transformed using expression vectors which may containviral origins of replication and/or endogenous expression elements and aselectable marker gene on the same or on a separate vector. Followingthe introduction of the vector, cells may be allowed to grow for 1-2days in an enriched media before they are switched to selective media.The purpose of the selectable marker is to confer resistance toselection, and its presence allows growth and recovery of cells whichsuccessfully express the introduced sequences. Resistant clones ofstably transformed cells may be proliferated using tissue culturetechniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) geneswhich can be employed in tk⁻ or aprt⁻ cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14) and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, β glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequence encoding HTRAN isinserted within a marker gene sequence, recombinant cells containingsequences encoding HTRAN can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding HTRAN under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

Alternatively, host cells which contain the nucleic acid sequenceencoding HTRAN and express HTRAN may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein.

The presence of polynucleotide sequences encoding HTRAN can be detectedby DNA-DNA or DNA-RNA hybridization or amplification using probes orportions or fragments of polynucleotides encoding HTRAN. Nucleic acidamplification based assays involve the use of oligonucleotides oroligomers based on the sequences encoding HTRAN to detect transformantscontaining DNA or RNA encoding HTRAN. As used herein "oligonucleotides"or "oligomers" refer to a nucleic acid sequence of at least about 10nucleotides and as many as about 60 nucleotides, preferably about 15 to30 nucleotides, and more preferably about 20-25 nucleotides, which canbe used as a probe or amplimer.

A variety of protocols for detecting and measuring the expression ofHTRAN, using either polyclonal or monoclonal antibodies specific for theprotein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson HTRAN is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding HTRAN includeoligolabeling, nick translation, end-labeling or PCR amplification usinga labeled nucleotide. Alternatively, the sequences encoding HTRAN, orany portions thereof may be cloned into a vector for the production ofan mRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits (Pharmacia & Upjohn, (Kalamazoo, Mich.);Promega (Madison Wis.); and U.S. Biochemical Corp., (Cleveland, Ohio)).Suitable reporter molecules or labels, which may be used, includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

Host cells transformed with nucleotide sequences encoding HTRAN may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a recombinantcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeHTRAN may be designed to contain signal sequences which direct secretionof HTRAN through a prokaryotic or eukaryotic cell membrane. Otherrecombinant constructions may be used to join sequences encoding HTRANto nucleotide sequence encoding a polypeptide domain which willfacilitate purification of soluble proteins. Such purificationfacilitating domains include, but are not limited to, metal chelatingpeptides such as histidine-tryptophan modules that allow purification onimmobilized metals, protein A domains that allow purification onimmobilized immunoglobulin, and the domain utilized in the FLAGSextension/affinity purification system (Immunex Corp., Seattle, Wash.).The inclusion of cleavable linker sequences such as those specific forFactor XA or enterokinase (Invitrogen, San Diego, Calif.) between thepurification domain and HTRAN may be used to facilitate purification.One such expression vector provides for expression of a fusion proteincontaining HTRAN and a nucleic acid encoding 6 histidine residuespreceding a thioredoxin or an enterokinase cleavage site. The histidineresidues facilitate purification on IMIAC (immobilized metal ionaffinity chromatography), as described in Porath, J. et al. (1992, Prot.Exp. Purif. 3: 263-281) while the enterokinase cleavage site provides ameans for purifying HTRAN from the fusion protein. A discussion ofvectors which contain fusion proteins is provided in Kroll, D. J. et al.(1993; DNA Cell Biol. 12:441-453).

In addition to recombinant production, fragments of HTRAN may beproduced by direct peptide synthesis using solid-phase techniques(Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesismay be performed using manual techniques or by automation. Automatedsynthesis may be achieved, for example, using an Applied Biosystems 431APeptide Synthesizer (Perkin Elmer). Various fragments of HTRAN may bechemically synthesized separately and combined using chemical methods toproduce the full length molecule.

THERAPEUTICS

Chemical and structural homology exists among HTRAN, mouse BKLF, andhuman BTEB2. In addition, northern analysis shows that cDNA librariescontaining HTRAN transcripts were from tumor-associated tissues andsynovial tissue of an arthritis patient. Thus, HTRAN expression appearsto be associated with cancer, arthritis, and developmental disorders.

HTRAN, a transcriptional activator, may be used to stimulate theexpression of genes that have a role in organ and organ systemdevelopment. Therefore, in one embodiment, HTRAN, a fragment, orderivative thereof, may be administered to a subject to treat or preventdevelopmental disorders, including but not limited to, renal tubularacidosis, anemia, Cushing's syndrome, achondroplastic dwarfism,epilepsy, gonadal dysgenesis, hereditary neuropathies such asCharcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism,hydrocephalus, seizure disorders such as Syndenham's chorea and cerebralpalsy, spinal bifida, and congenital glaucoma, cataract, orsensorineural hearing loss.

In another embodiment, a vector capable of expressing HTRAN, or afragment or a derivative thereof, may also be administered to a subjectto treat or prevent developmental disorders, including but not limitedto, the developmental disorders listed above.

Antagonists or inhibitors of HTRAN may also be used to suppresstranscriptional activation in arthritis patients. Thus in anotherembodiment, antagonists or inhibitors of HTRAN may be administered to asubject to treat or prevent arthritis.

In another embodiment, a vector expressing the complimentary sequence orantisense of the polynucleotide encoding HTRAN may be administered to asubject to treat or prevent arthritis.

Antagonists or inhibitors of HTRAN may be used to suppresstranscriptional activation in tumor cells. Thus in another embodiment,antagonists or inhibitors of HTRAN may be administered to a subject totreat or prevent cancer, including but not limited to, adenocarcinoma;leukemia; melanoma; lymphoma; sarcoma; and cancers of the bladder,colon, liver, brain, small intestine, large intestine, breast, ovary,kidney, lung, and prostate.

In another embodiment, a vector expressing the complimentary sequence orantisense of the polynucleotide encoding HTRAN may be administered to asubject to treat or prevent cancer. Examples of cancers include, but arenot limited to, the cancers listed above.

In other aspects, antibodies which are specific for HTRAN may be useddirectly as an antagonist, or indirectly as a targeting or deliverymechanism for bringing a pharmaceutical agent to cells or tissue whichexpress HTRAN.

In other embodiments, any of the therapeutic proteins, antagonists,antibodies, agonists, antisense sequences or vectors described above maybe administered in combination with other appropriate therapeuticagents. Selection of the appropriate agents for use in combinationtherapy may be made by one of ordinary skill in the art, according toconventional pharmaceutical principles. The combination of therapeuticagents may act synergistically to effect the treatment or prevention ofthe various disorders described above. Using this approach, one may beable to achieve therapeutic efficacy with lower dosages of each agent,thus reducing the potential for adverse side effects.

Antagonists or inhibitors of HTRAN may be produced using methods whichare generally known in the art. In particular, purified HTRAN may beused to produce antibodies or to screen libraries of pharmaceuticalagents to identify those which specifically bind HTRAN.

The antibodies may be generated using methods that are well known in theart. Such antibodies may include, but are not limited to, polyclonal,monoclonal, chimeric, single chain, Fab fragments, and fragmentsproduced by a Fab expression library. Neutralizing antibodies, (i.e.,those which inhibit dimer formation) are especially preferred fortherapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith HTRAN or any fragment or oligopeptide thereof which has immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include, but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

It is preferred that the peptides, fragments, or oligopeptides used toinduce antibodies to HTRAN have an amino acid sequence consisting of atleast five amino acids, and more preferably at least 10 amino acids. Itis also preferable that they are identical to a portion of the aminoacid sequence of the natural protein, and they may contain the entireamino acid sequence of a small, naturally occurring molecule. Shortstretches of HTRAN amino acids may be fused with those of anotherprotein such as keyhole limpet hemocyanin and antibody produced againstthe chimeric molecule.

Monoclonal antibodies to HTRAN may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. etal. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc.Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al. (1984) Mol. Cell Biol.62:109-120).

In addition, techniques developed for the production of "chimericantibodies", the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceHTRAN-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobulin libraries(Burton D. R. (1991) Proc. Natl. Acad. Sci. 88:11120-3).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inthe literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci.86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).

Antibody fragments which contain specific binding sites for HTRAN mayalso be generated. For example, such fragments include, but are notlimited to, the F(ab')2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab')2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 254:1275-1281).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between HTRAN and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering HTRAN epitopes is preferred, but a competitivebinding assay may also be employed (Maddox, supra).

In another embodiment of the invention, the polynucleotides encodingHTRAN, or any fragment thereof, or antisense molecules, may be used fortherapeutic purposes. In one aspect, antisense to the polynucleotideencoding HTRAN may be used in situations in which it would be desirableto block the transcription of the mRNA. In particular, cells may betransformed with sequences complementary to polynucleotides encodingHTRAN. Thus, antisense molecules may be used to modulate HTRAN activity,or to achieve regulation of gene function. Such technology is now wellknown in the art, and sense or antisense oligomers or larger fragments,can be designed from various locations along the coding or controlregions of sequences encoding HTRAN.

Expression vectors derived from retroviruses, adenovirus, herpes orvaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct recombinant vectors which will express antisensemolecules complementary to the polynucleotides of the gene encodingHTRAN. These techniques are described both in Sambrook et al. (supra)and in Ausubel et al. (supra).

Genes encoding HTRAN can be turned off by transforming a cell or tissuewith expression vectors which express high levels of a polynucleotide orfragment thereof which encodes HTRAN. Such constructs may be used tointroduce untranslatable sense or antisense sequences into a cell. Evenin the absence of integration into the DNA, such vectors may continue totranscribe RNA molecules until they are disabled by endogenousnucleases. Transient expression may last for a month or more with anon-replicating vector and even longer if appropriate replicationelements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning antisense molecules, DNA, RNA, or PNA, to the control regionsof the gene encoding HTRAN, i.e., the promoters, enhancers, and introns.Oligonucleotides derived from the transcription initiation site, e.g.,between positions -10 and +10 from the start site, are preferred.Similarly, inhibition can be achieved using "triple helix" base-pairingmethodology. Triple helix pairing is useful because it causes inhibitionof the ability of the double helix to open sufficiently for the bindingof polymerases, transcription factors, or regulatory molecules. Recenttherapeutic advances using triplex DNA have been described in theliterature (Gee, J. E. et al. (1994) In: Huber, B. E. and B. I. Carr,Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco,N.Y.). The antisense molecules may also be designed to block translationof mRNA by preventing the transcript from binding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding HTRAN.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Antisense molecules and ribozymes of the invention may be prepared byany method known in the art for the synthesis of nucleic acid molecules.These include techniques for chemically synthesizing oligonucleotidessuch as solid phase phosphoramidite chemical synthesis. Alternatively,RNA molecules may be generated by in vitro and in vivo transcription ofDNA sequences encoding HTRAN. Such DNA sequences may be incorporatedinto a wide variety of vectors with suitable RNA polymerase promoterssuch as T7 or SP6. Alternatively, these cDNA constructs that synthesizeantisense RNA constitutively or inducibly can be introduced into celllines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5' and/or 3' ends of the moleculeor the use of phosphorothioate or 2' O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection and by liposome injections may beachieved using methods which are well known in the art.

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical composition, in conjunction with a pharmaceuticallyacceptable carrier, for any of the therapeutic effects discussed above.Such pharmaceutical compositions may consist of HTRAN, antibodies toHTRAN, mimetics, agonists, antagonists, or inhibitors of HTRAN. Thecompositions may be administered alone or in combination with at leastone other agent, such as stabilizing compound, which may be administeredin any sterile, biocompatible pharmaceutical carrier, including, but notlimited to, saline, buffered saline, dextrose, and water. Thecompositions may be administered to a patient alone, or in combinationwith other agents, drugs or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Optionally, the suspensionmay also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tararic, malic, and succinic acids, etc. Saltstend to be more soluble in aqueous or other protonic solvents than arethe corresponding free base forms. In other cases, the preferredpreparation may be a lyophilized powder which may contain any or all ofthe following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, ata pH range of 4.5 to 5.5, that is combined with buffer prior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of HTRAN, such labeling would includeamount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually mice, rabbits, dogs, or pigs. The animal modelmay also be used to determine the appropriate concentration range androute of administration. Such information can then be used to determineuseful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example HTRAN or fragments thereof, antibodies of HTRAN,agonists, antagonists or inhibitors of HTRAN, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio of toxic to therapeutic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

DIAGNOSTICS

In another embodiment, antibodies which specifically bind HTRAN may beused for the diagnosis of conditions or diseases characterized byexpression of HTRAN, or in assays to monitor patients being treated withHTRAN, agonists, antagonists or inhibitors. The antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for HTRAN includemethods which utilize the antibody and a label to detect HTRAN in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

A variety of protocols including ELISA, RIA, and FACS for measuringHTRAN are known in the art and provide a basis for diagnosing altered orabnormal levels of HTRAN expression. Normal or standard values for HTRANexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody toHTRAN under conditions suitable for complex formation. The amount ofstandard complex formation may be quantified by various methods, butpreferably by photometric, means. Quantities of HTRAN expressed insubject, control and disease, samples from biopsied tissues are comparedwith the standard values. Deviation between standard and subject valuesestablishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingHTRAN may be used for diagnostic purposes. The polynucleotides which maybe used include oligonucleotide sequences, antisense RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofHTRAN may be correlated with disease. The diagnostic assay may be usedto distinguish between absence, presence, and excess expression ofHTRAN, and to monitor regulation of HTRAN levels during therapeuticintervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding HTRAN or closely related molecules, may be used to identifynucleic acid sequences which encode HTRAN. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5' regulatory region, or a less specific region,e.g., especially in the 3' coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding HTRAN, alleles, or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably contain at least 50% of the nucleotides from any ofthe HTRAN encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequence ofSEQ ID NO:2 or from genomic sequence including promoter, enhancerelements, and introns of the naturally occurring HTRAN.

Means for producing specific hybridization probes for DNAs encodingHTRAN include the cloning of nucleic acid sequences encoding HTRAN orHTRAN derivatives into vectors for the production of mRNA probes. Suchvectors are known in the art, commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, radionuclides such as 32P or 35S, or enzymatic labels, such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems, and the like.

Polynucleotide sequences encoding HTRAN may be used for the diagnosis ofconditions or diseases which are associated with expression of HTRAN.Examples of such conditions or diseases include arthritis and cancers ofthe bladder, colon, and lung. Additional examples of conditions ordiseases in which expression of HTRAN may be associated include: renaltubular acidosis; anemia; Cushing's syndrome; achondroplastic dwarfism;epilepsy; gonadal dysgenesis; hereditary neuropathies such asCharcot-Marie-Tooth disease and neurofibromatosis; hypothyroidism;hydrocephalus; seizure disorders such as Syndenham's chorea and cerebralpalsy; spinal bifida; congenital glaucoma; cataracts; sensorineuralhearing loss; adenocarcinoma; leukemia; melanoma; lymphoma; sarcoma; andcancers of the liver, brain, small intestine, large intestine, breast,ovary, kidney, and prostate. The polynucleotide sequences encoding HTRANmay be used in Southern or northern analysis, dot blot, or othermembrane-based technologies; in PCR technologies; or in dip stick, pIN,ELISA or chip assays utilizing fluids or tissues from patient biopsiesto detect altered HTRAN expression. Such qualitative or quantitativemethods are well known in the art.

In a particular aspect, the nucleotide sequences encoding HTRAN may beuseful in assays that detect activation or induction of various cancers,particularly those mentioned above. The nucleotide sequences encodingHTRAN may be labeled by standard methods, and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal in the biopsied or extracted sample issignificantly altered from that of a comparable control sample, thenucleotide sequences have hybridized with nucleotide sequences in thesample, and the presence of altered levels of nucleotide sequencesencoding HTRAN in the sample indicates the presence of the associateddisease. Such assays may also be used to evaluate the efficacy of aparticular therapeutic treatment regimen in animal studies, in clinicaltrials, or in monitoring the treatment of an individual patient.

In order to provide a basis for the diagnosis of disease associated withexpression of HTRAN, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, which encodes HTRAN, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withthose from an experiment where a known amount of a substantiallypurified polynucleotide is used. Standard values obtained from normalsamples may be compared with values obtained from samples from patientswho are symptomatic for disease. Deviation between standard and subjectvalues is used to establish the presence of disease.

Once disease is established and a treatment protocol is initiated,hybridization assays may be repeated on a regular basis to evaluatewhether the level of expression in the patient begins to approximatethat which is observed in the normal patient. The results obtained fromsuccessive assays may be used to show the efficacy of treatment over aperiod ranging from several days to months.

With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding HTRAN may involve the use of PCR. Such oligomers maybe chemically synthesized, generated enzymatically, or produced from arecombinant source. Oligomers will preferably consist of two nucleotidesequences, one with sense orientation (5'→3') and another with antisense(3'←5'), employed under optimized conditions for identification of aspecific gene or condition. The same two oligomers, nested sets ofoligomers, or even a degenerate pool of oligomers may be employed underless stringent conditions for detection and/or quantitation of closelyrelated DNA or RNA sequences.

Methods which may also be used to quantitate the expression of HTRANinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and standard curves onto which the experimentalresults are interpolated (Melby, P. C. et al. (1993) J. Immunol.Methods, 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem.212:229-236). The speed of quantitation of multiple samples may beaccelerated by running the assay in an ELISA format where the oligomerof interest is presented in various dilutions and a spectrophotometricor colorimetric response gives rapid quantitation.

In another embodiment of the invention, the nucleic acid sequences whichencode HTRAN may also be used to generate hybridization probes which areuseful for mapping the naturally occurring genomic sequence. Thesequences may be mapped to a particular chromosome or to a specificregion of the chromosome using well known techniques. Such techniquesinclude FISH, FACS, or artificial chromosome constructions, such asyeast artificial chromosomes, bacterial artificial chromosomes,bacterial P1 constructions or single chromosome cDNA libraries asreviewed in Price, C. M. (1993) Blood Rev. 7:127-134, and Trask, B. J.(1991) Trends Genet. 7:149-154.

FISH (as described in Verma et al. (1988) Human Chromosomes: A Manual ofBasic Techniques, Pergamon Press, New York, N.Y.) may be correlated withother physical chromosome mapping techniques and genetic map data.Examples of genetic map data can be found in the 1994 Genome Issue ofScience (265:1981f). Correlation between the location of the geneencoding HTRAN on a physical chromosomal map and a specific disease, orpredisposition to a specific disease, may help delimit the region of DNAassociated with that genetic disease. The nucleotide sequences of thesubject invention may be used to detect differences in gene sequencesbetween normal, carrier, or affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques such as linkage analysis using established chromosomalmarkers may be used for extending genetic maps. Often the placement of agene on the chromosome of another mammalian species, such as mouse, mayreveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms, or parts thereof, by physical mapping. This providesvaluable information to investigators searching for disease genes usingpositional cloning or other gene discovery techniques. Once the diseaseor syndrome has been crudely localized by genetic linkage to aparticular genomic region, for example, AT to 11q22-23 (Gatti, R. A. etal. (1988) Nature 336:577-580), any sequences mapping to that area mayrepresent associated or regulatory genes for further investigation. Thenucleotide sequence of the subject invention may also be used to detectdifferences in the chromosomal location due to translocation, inversion,etc. among normal, carrier, or affected individuals.

In another embodiment of the invention, HTRAN, and catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, betweenHTRAN and the agent being tested, may be measured.

Another technique for drug screening which may be used provides for highthroughput screening of compounds having suitable binding affinity tothe protein of interest as described in published PCT applicationWO84/03564. In this method, as applied to HTRAN large numbers ofdifferent small test compounds are synthesized on a solid substrate,such as plastic pins or some other surface. The test compounds arereacted with HTRAN, or fragments thereof, and washed. Bound HTRAN isthen detected by methods well known in the art. Purified HTRAN can alsobe coated directly onto plates for use in the aforementioned drugscreening techniques. Alternatively, non-neutralizing antibodies can beused to capture the peptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding HTRAN specificallycompete with a test compound for binding HTRAN. In this manner, theantibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with HTRAN.

In additional embodiments, the nucleotide sequences which encode HTRANmay be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES

I SYNOOAT01 cDNA Library Construction

The osteoarthritic knee joint from a 82 year-old female used for cDNAlibrary construction was obtained from the University of CaliforniaDavis. The frozen tissue was homogenized using a Brinkmann HomogenizerPolytron PT-3000 (Brinkmann Instruments, Westbury N.J.) and lysed in abuffer containing guanidinium isothiocyanate. The lysate was centrifugedover a 5.7 M CsCl cushion using an Beckman SW28 rotor in a BeckmanL8-70M Ultracentrifuge (Beckman Instruments) for 18 hours at 25,000 rpmat ambient temperature. The RNA was extracted twice with acid phenol pH4.0 using the reagents and extraction procedures as supplied in theStratagene RNA Isolation Kit (Catalog #200345; Stratagene). RNA wasprecipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol,resuspended in water and DNase treated for 15 min at 37° C. The RNA wasisolated using the Qiagen Oligotex kit (QIAGEN Inc, Chatsworth Calif.).

The poly-A⁺ RNA was handled according to the recommended protocols inthe SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning(Catalog #8248-013; Gibco/BRL). First strand cDNA synthesis wasaccomplished using oligo d(T) priming and second strand synthesis wasperformed using a combination of DNA polymerase I, E. coli ligase andRNase H. The cDNA was blunted with T4 polymerase, and a Sal I linker wasadded to the blunt ended cDNA. The Sal I adapted, double-stranded cDNAswere the digested with Not I and fractionated on a Sepharose CL4B column(Catalog #275105, Pharmacia). Those cDNAs exceeding 400 bp were ligatedinto pSport I which was subsequently transformed into DH5a™ competentcells (Catalog #18258-012, Gibco/BRL).

II Isolation and Sequencing of cDNA Clones

Plasmid DNA was released from the cells and purified using the MiniprepKit (Catalog #77468; Advanced Genetic Technologies Corporation,Gaithersburg Md.). This kit consists of a 96-well block with reagentsfor 960 purifications. The recommended protocol was employed except forthe following changes: 1) the 96 wells were each filled with only 1 mlof sterile Terrific Broth (Catalog #22711, LIFE TECHNOLOGIES™) withcarbenicillin at 25 mg/L and glycerol at 0.4%; 2) the bacteria werecultured for 24 hours after the wells were inoculated and then lysedwith 60 μl of lysis buffer; 3) a centrifugation step employing theBeckman GS-6R rotor at 2900 rpm for 5 minutes was performed before thecontents of the block were added to the primary filter plate; and 4) theoptional step of adding isopropanol to TRIS buffer was not routinelyperformed. After the last step in the protocol, samples were transferredto a Beckman 96-well block for storage.

The cDNAs were sequenced by the method of Sanger F and A R Coulson(1975; J Mol Biol 94:441f), using a Hamilton Micro Lab 2200 (Hamilton,Reno Nev.) in combination with four Peltier Thermal Cyclers (PTC200 fromMJ Research, Watertown Mass.) and Applied Biosystems 377 or 373 DNASequencing Systems, and the reading frame was determined.

III Homology Searching of cDNA Clones and Their Deduced Proteins

The nucleotide sequences of the Sequence Listing or amino acid sequencesdeduced from them were used as query sequences against databases such asGenBank, SwissProt, BLOCKS, and Pima II. These databases which containpreviously identified and annotated sequences were searched for regionsof homology (similarity) using BLAST, which stands for Basic LocalAlignment Search Tool (Altschul (1993) supra, Altschul (1990) supra).

BLAST produces alignments of both nucleotide and amino acid sequences todetermine sequence similarity. Because of the local nature of thealignments, BLAST is especially useful in determining exact matches orin identifying homologs which may be of prokaryotic (bacterial) oreukaryotic (animal, fungal or plant) origin. Other algorithms such asthe one described in Smith R F and T F Smith (1992 Protein Engineering5:35-51), incorporated herein by reference, can be used when dealingwith primary sequence patterns and secondary structure gap penalties. Asdisclosed in this application, the sequences have lengths of at least 49nucleotides, and no more than 12% uncalled bases (where N is recordedrather than A, C, G, or T).

The BLAST approach, as detailed in Karlin and Altschul (supra) andincorporated herein by reference, searches matches between a querysequence and a database sequence, to evaluate the statisticalsignificance of any matches found, and to report only those matcheswhich satisfy the user-selected threshold of significance. In thisapplication, threshold was set at 10⁻²⁵ for nucleotides and 10⁻¹⁴ forpeptides.

Incyte nucleotide sequence were searched against the GenBank databasesfor primate (pri), rodent (rod), and mammalian sequences (mam), anddeduced amino acid sequences from the same clones are searched againstGenBank functional protein databases, mammalian (mamp), vertebrate(vrtp) and eukaryote (eukp), for homology. The relevant database for aparticular match were reported as a GIxxx±p (where xxx is pri, rod, etcand if present, p=peptide). The product score is calculated as follows:the % nucleotide or amino acid identity [between the query and referencesequences] in BLAST is multiplied by the % maximum possible BLAST score[based on the lengths of query and reference sequences] and then dividedby 100. Where an Incyte Clone was homologous to several sequences, up tofive matches were provided with their relevant scores. In an analogy tothe hybridization procedures used in the laboratory, the electronicstringency for an exact match was set at 70, and the conservative lowerlimit for an exact match was set at approximately 40 (with 1-2% errordue to uncalled bases).

IV Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound (Sambrook et al., supra).

Analogous computer techniques using BLAST (Altschul, S. F. 1993 and1990, supra) are used to search for identical or related molecules innucleotide databases such as GenBank or the LIFESEQ™ database (IncytePharmaceuticals). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

The basis of the search is the product score which is defined as:

    % sequence identity×% maximum BLAST score/100

The product score takes into account both the degree of similaritybetween two sequences and the length of the sequence match. For example,with a product score of 40, the match will be exact within a 1-2% error;and at 70, the match will be exact. Homologous molecules are usuallyidentified by selecting those which show product scores between 15 and40, although lower scores may identify related molecules.

The results of northern analysis are reported as a list of libraries inwhich the transcript encoding HTRAN occurs. Abundance and percentabundance are also reported. Abundance directly reflects the number oftimes a particular transcript is represented in a cDNA library, andpercent abundance is abundance divided by the total number of sequencesexamined in the cDNA library.

V Extension of HTRAN-Encoding Polynucleotides

Incyte clone 820694 or HTRAN-encoding nucleic acid sequence (SEQ IDNO:2) is used to design oligonucleotide primers for extending a partialnucleotide sequence to full length or for obtaining 5' or 3', intron orother control sequences from genomic libraries. One primer issynthesized to initiate extension in the antisense direction (XLR) andthe other is synthesized to extend sequence in the sense direction(XLF). Primers are used to facilitate the extension of the knownsequence "outward" generating amplicons containing new, unknownnucleotide sequence for the region of interest. The initial primers aredesigned from the cDNA using OLIGO 4.06 (National Biosciences), oranother appropriate program, to be 22-30 nucleotides in length, to havea GC content of 50% or more, and to anneal to the target sequence attemperatures about 68°-72° C. Any stretch of nucleotides which wouldresult in hairpin structures and primer-primer dimerizations is avoided.

The original, selected cDNA libraries, or a human genomic library areused to extend the sequence; the latter is most useful to obtain 5'upstream regions. If more extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

By following the instructions for the XL-PCR kit (Perkin Elmer) andthoroughly mixing the enzyme and reaction mix, high fidelityamplification is obtained. Beginning with 40 pmol of each primer and therecommended concentrations of all other components of the kit, PCR isperformed using the Peltier Thermal Cycler (PTC200; M.J. Research,Watertown, Mass.) and the following parameters:

    ______________________________________                                        Step 1       94° C. for 1 min (initial denaturation)                                 Step 2 65° C. for 1 min                                    Step 3 68° C. for 6 min                                                Step 4 94° C. for 15 sec                                               Step 5 65° C. for 1 min                                                Step 6 68° C. for 7 min                                                Step 7 Repeat step 4-6 for 15 additional cycles                               Step 8 94° C. for 15 sec                                               Step 9 65° C. for 1 min                                                Step 10 68° C. for 7:15 min                                            Step 11 Repeat step 8-10 for 12 cycles                                        Step 12 72° C. for 8 min                                               Step 13 4° C. (and holding)                                          ______________________________________                                    

A 5-10 μl aliquot of the reaction mixture is analyzed by electrophoresison a low concentration (about 0.6-0.8%) agarose mini-gel to determinewhich reactions were successful in extending the sequence. Bands thoughtto contain the largest products are selected and removed from the gel.Further purification involves using a commercial gel extraction methodsuch as QIAQuick™ (QIAGEN Inc., Chatsworth, Calif.). After recovery ofthe DNA, Klenow enzyme is used to trim single-stranded, nucleotideoverhangs creating blunt ends which facilitate religation and cloning.

After ethanol precipitation, the products are redissolved in 13 μl ofligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase are added, and the mixture is incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) are transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C., the whole transformationmixture is plated on Luria Bertani (LB)-agar (Sambrook et al., supra)containing 2x Carb. The following day, several colonies are randomlypicked from each plate and cultured in 150 μl of liquid LB/2x Carbmedium placed in an individual well of an appropriate,commercially-available, sterile 96-well microtiter plate. The followingday, 5 μl of each overnight culture is transferred into a non-sterile96-well plate and after dilution 1:10 with water, 5 μl of each sample istransferred into a PCR array.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×)containing 4 units of rTth DNA polymerase, a vector primer, and one orboth of the gene specific primers used for the extension reaction areadded to each well. Amplification is performed using the followingconditions:

    ______________________________________                                        Step 1     94° C. for 60 sec                                             Step 2 94° C. for 20 sec                                               Step 3 55° C. for 30 sec                                               Step 4 72° C. for 90 sec                                               Step 5 Repeat steps 2-4 for an additional 29 cycles                           Step 6 72° C. for 180 sec                                              Step 7 4° C. (and holding)                                           ______________________________________                                    

Aliquots of the PCR reactions are run on agarose gels together withmolecular weight markers. The sizes of the PCR products are compared tothe original partial cDNAs, and appropriate clones are selected, ligatedinto plasmid, and sequenced.

VI Labeling and Use of Hybridization Probes

Hybridization probes derived from SEQ ID NO:2 are employed to screencDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base-pairs, is specificallydescribed, essentially the same procedure is used with larger cDNAfragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 (National Biosciences), and labeled by combining 50pmol of each oligomer and 250 μCi of [γ-³² P] adenosine triphosphate(Amersham) and T4 polynucleotide kinase (DuPont NEN®, Boston, Mass.).The labeled oligonucleotides are substantially purified with SephadexG-25 superfine resin column (Pharmacia & Upjohn). A portion containing10⁷ counts per minute of each of the sense and antisenseoligonucleotides is used in a typical membrane based hybridizationanalysis of human genomic DNA digested with one of the followingendonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1, or Pvu II; DuPontNEN®).

The DNA from each digest is fractionated on a 0.7 percent agarose geland transferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham, N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR™ film(Kodak, Rochester, N.Y.) is exposed to the blots in a Phosphoimagercassette (Molecular Dynamics, Sunnyvale, Calif.), hybridization patternsare compared visually.

VII Antisense Molecules

Antisense molecules to the HTRAN-encoding sequence, or any part thereof,is used to inhibit in vivo or in vitro expression of naturally occurringHTRAN. Although use of antisense oligonucleotides, comprising about 20base-pairs, is specifically described, essentially the same procedure isused with larger cDNA fragments. An oligonucleotide based on the codingsequences of HTRAN, as shown in FIGS. 1A, 1B, 1C, 1D, and 1E, is used toinhibit expression of naturally occurring HTRAN. The complementaryoligonucleotide is designed from the most unique 5' sequence as shown inFIGS. 1A, 1B, 1C, 1D, and 1E and used either to inhibit transcription bypreventing promoter binding to the upstream nontranslated sequence ortranslation of an HTRAN-encoding transcript by preventing the ribosomefrom binding. Using an appropriate portion of the signal and 5' sequenceof SEQ ID NO:2, an effective antisense oligonucleotide includes any15-20 nucleotides spanning the region which translates into the signalor 5' coding sequence of the polypeptide as shown in FIGS. 1A, 1B, 1C,1D, and 1E.

VIII Expression of HTRAN

Expression of HTRAN is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector, pSport, previously used for thegeneration of the cDNA library is used to express HTRAN in E. coli.Upstream of the cloning site, this vector contains a promoter forβ-galactosidase, followed by sequence containing the amino-terminal Met,and the subsequent seven residues of β-galactosidase. Immediatelyfollowing these eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

Induction of an isolated, transformed bacterial strain with IPTG usingstandard methods produces a fusion protein which consists of the firsteight residues of β-galactosidase, about 5 to 15 residues of linker, andthe full length protein. The signal residues direct the secretion ofHTRAN into the bacterial growth media which can be used directly in thefollowing assay for activity.

IX Demonstration of HTRAN Activity

The binding of Zn²⁺ to HTRAN is assayed by monitoring the resultingchanges in enthalpy (heat production or absorption) in an isothermaltitration microcalorimeter (Micro-Cal Inc., Northampton, Mass.).Titration microcalorimetry measurements do not require labeling of theligand or receptor molecules; detection is based solely on the intrinsicchange in the heat of enthalpy upon binding. Multiplecomputer-controlled injections of a known volume of ZnCl₂ solution aredirected into a thermally-controlled chamber containing HTRAN. Thechange in enthalpy after each injection is plotted against the number ofinjections, producing a binding isotherm. The volumes and concentrationsof the injected ZnCl₂ solution and of the HTRAN solution are used alongwith the binding isotherm to calculate values for the number, affinity,and association constant of Zn²⁺ with HTRAN.

X Production of HTRAN Specific Antibodies

HTRAN that is substantially purified using PAGE electrophoresis(Sambrook, supra), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols. The aminoacid sequence deduced from SEQ ID NO:2 is analyzed using DNASTARsoftware (DNASTAR Inc) to determine regions of high immunogenicity and acorresponding oligopolypeptide is synthesized and used to raiseantibodies by means known to those of skill in the art. Selection ofappropriate epitopes, such as those near the C-terminus or inhydrophilic regions, is described by Ausubel et al. (supra), and others.

Typically, the oligopeptides are 15 residues in length, synthesizedusing an Applied Biosystems Peptide Synthesizer Model 431A usingfmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH, Sigma,St. Louis, Mo.) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimideester (MBS; Ausubel et al., supra). Rabbits are immunized with theoligopeptide-KLH complex in complete Freund's adjuvant. The resultingantisera are tested for antipeptide activity, for example, by bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radioiodinated, goat anti-rabbitIgG.

XI Purification of Naturally Occurring HTRAN Using Specific Antibodies

Naturally occurring or recombinant HTRAN is substantially purified byimmunoaffinity chromatography using antibodies specific for HTRAN. Animmnunoaffinity column is constructed by covalently coupling HTRANantibody to an activated chromatographic resin, such as CnBr-activatedSepharose (Pharmacia & Upjohn). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

Media containing HTRAN is passed over the immunoaffinity column, and thecolumn is washed under conditions that allow the preferential absorbanceof HTRAN (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/HTRAN binding (eg, a buffer of pH 2-3 or a high concentrationof a chaotrope, such as urea or thiocyanate ion), and HTRAN iscollected.

XII Identification of Molecules Which Interact with HTRAN

HTRAN or biologically active fragments thereof are labeled with ¹²⁵ IBolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133: 529).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled HTRAN, washed and any wells withlabeled HTRAN complex are assayed. Data obtained using differentconcentrations of HTRAN are used to calculate values for the number,affinity, and association of HTRAN with the candidate molecules.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 4                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 345 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: SYN00AT01                                                        (B) CLONE: 727885                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - -  Met Leu Met Phe Asp Pro Val Pro Val Xaa - #Gln Glu Ala Met Asp        Pro                                                                               1               5 - #                 10 - #                 15             - -  Val Ser Val Ser Tyr Pro Ser Asn Tyr Met - #Glu Ser Met Lys Pro Asn                   20     - #             25     - #             30                  - -  Lys Tyr Gly Val Ile Tyr Ser Thr Pro Leu - #Pro Glu Lys Phe Phe Gln               35         - #         40         - #         45                      - -  Thr Pro Glu Gly Leu Ser His Gly Ile Gln - #Met Glu Pro Val Asp Leu           50             - #     55             - #     60                          - -  Thr Val Asn Lys Arg Ser Ser Pro Pro Ser - #Ala Gly Asn Ser Pro Ser       65                 - # 70                 - # 75                 - # 80       - -  Ser Leu Lys Phe Pro Ser Ser His Arg Arg - #Ala Ser Pro Gly Leu Ser                       85 - #                 90 - #                 95              - -  Met Pro Ser Ser Ser Pro Pro Ile Lys Lys - #Tyr Ser Pro Pro Ser Pro                   100     - #            105     - #            110                 - -  Gly Val Gln Pro Phe Gly Val Pro Leu Ser - #Met Pro Pro Val Met Ala               115         - #        120         - #        125                     - -  Ala Ala Leu Ser Arg His Gly Ile Arg Ser - #Pro Gly Ile Leu Pro Val           130             - #    135             - #    140                         - -  Ile Gln Pro Val Val Val Gln Pro Val Pro - #Phe Met Tyr Thr Ser His       145                 - #150                 - #155                 -         #160                                                                             - -  Leu Gln Gln Pro Leu Met Val Ser Leu Ser - #Glu Glu Met Glu Asn        Ser                                                                                              165 - #                170 - #                175            - -  Ser Ser Ser Met Gln Val Pro Val Ile Glu - #Ser Tyr Glu Lys Pro Ile                   180     - #            185     - #            190                 - -  Ser Gln Lys Lys Ile Lys Ile Glu Pro Gly - #Ile Glu Pro Gln Arg Thr               195         - #        200         - #        205                     - -  Asp Tyr Tyr Pro Glu Glu Met Ser Pro Pro - #Leu Met Asn Ser Val Ser           210             - #    215             - #    220                         - -  Pro Pro Gln Ala Leu Leu Gln Glu Asn His - #Pro Ser Val Ile Val Gln       225                 - #230                 - #235                 -         #240                                                                             - -  Pro Gly Lys Arg Pro Leu Pro Val Glu Ser - #Pro Asp Thr Gln Arg        Lys                                                                                              245 - #                250 - #                255            - -  Arg Arg Ile His Arg Cys Asp Tyr Asp Gly - #Cys Asn Lys Val Tyr Thr                   260     - #            265     - #            270                 - -  Lys Ser Ser His Leu Lys Ala His Arg Arg - #Thr His Thr Gly Glu Lys               275         - #        280         - #        285                     - -  Pro Tyr Lys Cys Thr Trp Glu Gly Cys Thr - #Trp Lys Phe Ala Arg Ser           290             - #    295             - #    300                         - -  Asp Glu Leu Thr Arg His Phe Arg Lys His - #Thr Gly Ile Lys Pro Phe       305                 - #310                 - #315                 -         #320                                                                             - -  Gln Cys Pro Asp Cys Asp Arg Ser Phe Ser - #Arg Ser Asp His Leu        Ala                                                                                              325 - #                330 - #                335            - -  Leu His Arg Lys Arg His Met Leu Val                                                  340     - #            345                                        - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1496 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: SYNOOAT01                                                        (B) CLONE: 727885                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - -  CCTCGCAAAN CCNAACACCA AAGCANCTAG GAAGGTTTAA CTAAAAGAAT - #GCTCATGTT    T    60                                                                         - -  GACCCAGTTC CTGTCAANCA AGAGGCCATG GACCCTGTCT CAGTGTCATA - #CCCATCTAA    T   120                                                                         - -  TACATGGAAT CCATGAAGCC TAACAAGTAT GGGGTCATCT ACTCCACACC - #ATTGCCTGA    G   180                                                                         - -  AAGTTCTTTC AGACCCCAGA AGGTCTGTCG CACGGAATAC AGATGGAGCC - #AGTGGACCT    C   240                                                                         - -  ACGGTGAACA AGCGGAGTTC ACCCCCTTCG GCTGGGAATT CGCCCTCCTC - #TCTGAAGTT    C   300                                                                         - -  CCGTCCTCAC ACCGGAGAGC CTCGCCTGGG TTGAGCATGC CTTCTTCCAG - #CCCACCGAT    A   360                                                                         - -  AAAAAATACT CACCCCCTTC TCCAGGCGTG CAGCCCTTCG GCGTGCCGCT - #GTCCATGCC    A   420                                                                         - -  CCAGTGATGG CAGCTGCCCT CTCGCGGCAT GGAATACGGA GCCCGGGGAT - #CCTGCCCGT    C   480                                                                         - -  ATCCAGCCGG TGGTGGTGCA GCCCGTCCCC TTTATGTACA CAAGTCACCT - #CCAGCAGCC    T   540                                                                         - -  CTCATGGTCT CCTTATCGGA GGAGATGGAA AATTCCAGTA GTAGCATGCA - #AGTACCTGT    A   600                                                                         - -  ATTGAATCAT ATGAGAAGCC TATATCACAG AAAAAAATTA AAATAGAACC - #TGGGATCGA    A   660                                                                         - -  CCACAGAGGA CAGATTATTA TCCTGAAGAA ATGTCACCCC CCTTAATGAA - #CTCAGTGTC    C   720                                                                         - -  CCCCCGCAAG CATTGTTGCA AGAGAATCAC CCTTCGGTCA TCGTGCAGCC - #TGGGAAGAG    A   780                                                                         - -  CCTTTACCTG TGGAATCCCC GGATACTCAA AGGAAGCGGA GGATACACAG - #ATGTGATTA    T   840                                                                         - -  GATGGATGCA ACAAAGTGTA CACTAAAAGC TCCCACTTGA AAGCACACAG - #AAGAACACA    C   900                                                                         - -  ACAGGAGAAA AACCCTACAA ATGTACATGG GAAGGGTGCA CATGGAAGTT - #TGCTCGGTC    T   960                                                                         - -  GATGAACTAA CAAGACATTT CCGAAAACAT ACTGGAATCA AACCTTTCCA - #GTGCCCGGA    C  1020                                                                         - -  TGTGACCGCA GCTTCTCCCG TTCTGACCAT CTTGCCCTCC ATAGGAAACG - #CCACATGCT    A  1080                                                                         - -  GTCTGATTGC CTCTGTGTCC TGCCTCAGCG TGACTCCCCA CTCACCTGGC - #TCTCTCTCT    G  1140                                                                         - -  TCCTGCCTCC CATTATCTAA CACATTTTTT ACATGTACAT TTTAATTTGA - #TTCAGCTGG    T  1200                                                                         - -  CTGAATCTCT GAATTTATAT CATCCAAAAC TTCCATATGG TCAGTAGTAG - #ATGTTCTCT    A  1260                                                                         - -  ATCCTCCCTC TCCTTACCAC GGGTCAGACC TAAAGAATGT GAACACTTTT - #TTTTTTTTT    T  1320                                                                         - -  CTGGGGATGC TAAGCAAACC CTTCTTACAG ATACGTTTAA TGTTATAAGG - #AACAAGGGA    A  1380                                                                         - -  CNTGTNAACT AACATAACCA ATTGTCAGTT CTCCNTGTAT TCCTCAAAAG - #AATGTCAAA    A  1440                                                                         - -  NTAAATGTTT TAAAAATCNA CACCTCAANN NCAAAAAAAN ANNANTTAAT - #AAAAGG          1496                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 344 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: GenBank                                                          (B) CLONE: 1244515                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - -  Met Leu Met Phe Asp Pro Val Pro Val Lys - #Gln Glu Ala Met Asp Pro        1               5 - #                 10 - #                 15              - -  Val Ser Val Ser Phe Pro Ser Asn Tyr Ile - #Glu Ser Met Lys Pro Asn                   20     - #             25     - #             30                  - -  Lys Tyr Gly Val Ile Tyr Ser Thr Pro Leu - #Pro Asp Lys Phe Phe Gln               35         - #         40         - #         45                      - -  Thr Pro Glu Gly Leu Thr His Gly Ile Gln - #Val Glu Pro Val Asp Leu           50             - #     55             - #     60                          - -  Thr Val Asn Lys Arg Gly Ser Pro Pro Ala - #Ala Gly Gly Ser Pro Ser       65                 - # 70                 - # 75                 - # 80       - -  Ser Leu Lys Phe Pro Ser His Arg Arg Ala - #Ser Pro Gly Leu Ser Met                       85 - #                 90 - #                 95              - -  Pro Ser Ser Ser Pro Pro Ile Lys Lys Tyr - #Ser Pro Pro Ser Pro Gly                   100     - #            105     - #            110                 - -  Val Gln Pro Phe Gly Val Pro Leu Ser Met - #Pro Pro Val Met Ala Ala               115         - #        120         - #        125                     - -  Ala Leu Ser Arg His Gly Ile Arg Ser Pro - #Gly Ile Leu Pro Val Ile           130             - #    135             - #    140                         - -  Gln Pro Val Val Val Gln Pro Val Pro Phe - #Met Tyr Thr Ser His Leu       145                 - #150                 - #155                 -         #160                                                                             - -  Gln Gln Pro Leu Met Val Ser Leu Ser Glu - #Glu Met Asp Asn Ser        Asn                                                                                              165 - #                170 - #                175            - -  Ser Gly Met Pro Val Pro Val Ile Glu Ser - #Tyr Glu Lys Pro Leu Leu                   180     - #            185     - #            190                 - -  Gln Lys Lys Ile Lys Ile Glu Pro Gly Ile - #Glu Pro Gln Arg Thr Asp               195         - #        200         - #        205                     - -  Tyr Tyr Pro Glu Glu Met Ser Pro Pro Leu - #Met Asn Pro Val Ser Pro           210             - #    215             - #    220                         - -  Pro Gln Ala Leu Leu Gln Glu Asn His Pro - #Ser Val Ile Val Gln Pro       225                 - #230                 - #235                 -         #240                                                                             - -  Gly Lys Arg Pro Leu Pro Val Glu Ser Pro - #Asp Thr Gln Arg Lys        Arg                                                                                              245 - #                250 - #                255            - -  Arg Ile His Arg Cys Asp Tyr Asp Gly Cys - #Asn Lys Val Tyr Thr Lys                   260     - #            265     - #            270                 - -  Ser Ser His Leu Lys Ala His Arg Arg Thr - #His Thr Gly Glu Lys Pro               275         - #        280         - #        285                     - -  Tyr Lys Cys Thr Trp Glu Gly Cys Thr Trp - #Lys Phe Ala Arg Ser Asp           290             - #    295             - #    300                         - -  Glu Leu Thr Arg His Phe Arg Lys His Thr - #Gly Ile Lys Pro Phe Gln       305                 - #310                 - #315                 -         #320                                                                             - -  Cys Pro Asp Cys Asp Arg Ser Phe Ser Arg - #Ser Asp His Leu Ala        Leu                                                                                              325 - #                330 - #                335            - -  His Arg Lys Arg His Met Leu Val                                                      340                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 219 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: GenBank                                                          (B) CLONE: 303597                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - -  Met Pro Ser Ser Thr Asn Gln Thr Ala Ala - #Met Asp Thr Leu Asn Val        1               5 - #                 10 - #                 15              - -  Ser Met Ser Ala Ala Met Ala Gly Leu Asn - #Thr His Thr Ser Ala Val                   20     - #             25     - #             30                  - -  Pro Gln Thr Ala Val Lys Gln Phe Gln Gly - #Met Pro Pro Cys Thr Tyr               35         - #         40         - #         45                      - -  Thr Met Pro Ser Gln Phe Leu Pro Gln Gln - #Ala Thr Tyr Phe Pro Pro           50             - #     55             - #     60                          - -  Ser Pro Pro Ser Ser Glu Pro Gly Ser Pro - #Asp Arg Gln Ala Glu Met       65                 - # 70                 - # 75                 - # 80       - -  Leu Gln Asn Leu Thr Pro Pro Pro Ser Tyr - #Ala Ala Thr Ile Ala Ser                       85 - #                 90 - #                 95              - -  Lys Leu Ala Ile His Asn Pro Asn Leu Pro - #Thr Thr Leu Pro Val Asn                   100     - #            105     - #            110                 - -  Ser Gln Asn Ile Gln Pro Val Arg Tyr Asn - #Arg Arg Ser Asn Pro Asp               115         - #        120         - #        125                     - -  Leu Glu Lys Arg Arg Ile His Tyr Cys Asp - #Tyr Pro Gly Cys Thr Lys           130             - #    135             - #    140                         - -  Val Tyr Thr Lys Ser Ser His Leu Lys Ala - #His Leu Arg Thr His Thr       145                 - #150                 - #155                 -         #160                                                                             - -  Gly Glu Lys Pro Tyr Lys Cys Thr Trp Glu - #Gly Cys Asp Trp Arg        Phe                                                                                              165 - #                170 - #                175            - -  Ala Arg Ser Asp Glu Leu Thr Arg His Tyr - #Arg Lys His Thr Gly Ala                   180     - #            185     - #            190                 - -  Lys Pro Phe Gln Cys Gly Val Cys Asn Arg - #Ser Phe Ser Arg Ser Asp               195         - #        200         - #        205                     - -  His Leu Ala Leu His Met Lys Arg His Gln - #Asn                               210             - #    215                                              __________________________________________________________________________

What is claimed is:
 1. A method for detecting a polynucleotide which mayencode an HTRAN (human transcription factor) polypeptide in a biologicalsample, comprising the steps of:a) incubating under stringenthybridization conditions a polynucleotide comprising SEQ ID NO:2 and apolynucleotide in the biological sample; and b) detecting anyhybridization complex which forms between said polynucleotide comprisingSEQ ID NO:2 and said polynucleotide in the biological sample, whereinthe detection of said complex indicates that said polynucleotide in thebiological sample may encode said HTRAN polypeptide.